The invention relates generally to interferometric microscopy including, for example, interferometric microscopy of samples comprised of or labeled with spatially resolvable point sources such as samples that internally radiate light from photo-activated fluorescent protein molecules and/or interferometric microscopy where a phase difference is measured to determine position information in the third dimension (e.g., the z coordinate) while simultaneously measuring position information in the two other dimensions (e.g., the x and y coordinates).
Microscopy systems that measure interferometric phase are known. For example, one known microscopy system measures interferometric phase by illuminating an extended surface of an object with an external light source. Light reflected from the sample interferes with a reference beam in a beam splitter. The interfered beam is imaged onto a detector. The optical path difference between the reference beam and the sample beam can modulate the amplitude of the output interfered beam and provide a measure of an object height.
Also known are microscopy systems that interfere two beams from a sample via opposing objectives to measure only one interfered output beam. Such microscopy systems do not explicitly measure phase. These microscopy systems can resolve structure in the axial dimension.
Such known microscopy systems, however, suffer several disadvantages. For example, such systems cannot be applied to fluorescently-labeled samples that are common in biology because the internally-supplied fluorescent radiation has no useable phase relation with respect to any externally-supplied reference beam used for excitation of fluorescence. Without a reference beam, interference is not achievable. In addition, a single or sequentially phased interferometric measurement cannot be used to separate interferometric phase from amplitude if the radiation is transient or strongly time variant, such as is the case in blinking fluorescent molecules or other single photon sources. Thus, no quantitative measure of phase and positional information can be derived from a single interferometric measurement or a time sequence of measurements. Finally, lateral resolution for far-field interferometric systems is limited by the Abbe diffraction length λ/2NA, where λ is the wavelength and NA is the numerical aperture. Thus, a need exists for an improved microscopy system.